Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells
Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells
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DOI：
10.1074/jbc.275.14.10648
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发表时间：
2000-04-07
影响因子：
4.8
通讯作者：
Jeunemaitre, X
中科院分区：
文献类型：
--
作者：
Célérier, J;Schmid, G;Jeunemaitre, X
血管紧张素原、肾素（E.C. 3.4.23.15）底物，属于丝氨酸蛋白酶抑制剂超家族，并已被归类为非抑制性丝氨酸蛋白酶抑制剂。在整个纯化过程中不存在蛋白酶抑制剂导致血管紧张素原在反应中心环内裂解，这种裂解不影响Ang I的产生，因为动力学参数与全长血管紧张素原的值相似。虽然切割是完全的，但切割的血管紧张素原在去糖基化后在SDS-聚丙烯酰胺凝胶电泳上迁移为一个相差4 kDa的双联体。为了测试循环血管紧张素原是否在反应中心环中被切割，从人血浆池中纯化并显示其未被切割。它的迁移明显慢于切割的血管紧张素原，但也包括两条带，指向迄今无法解释的残余异质性。然后，我们比较了全长和反应中心环裂解血管紧张素原的热诱导聚合。这两个单体都能够聚集，揭示了一个特殊的行为，血管紧张素原不同的反应中心环裂解丝氨酸蛋白酶抑制剂，缺乏血管紧张素原的三维结构，我们提出并讨论了结构模型的丝氨酸蛋白酶抑制剂折叠内的肾素基板。
Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin, Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop, This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length-and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins, Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.